Chromone–lipoic Acid Conjugate: Neuroprotective Agent Having Acceptable Butyrylcholinesterase Inhibition, Antioxidant And Copper-chelation Activities

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Abstract

Purpose Alzheimer’s disease (AD) is a multifaceted neurodegenerative disease. To target simultaneously multiple pathological processes involved in Alzheimer's Disease, natural-origin compounds with unique characteristics are promising scaffolds to develop novel multi-target compounds in the treatment of different neurodegenerative diseases, especially Alzheimer's Disease. In this study, novel chromone-lipoic acid hybrids were prepared to find a new multifunctional lead structure for the treatment of AD. Methods Chromone-lipoic acid hybrids were prepared through click reaction and their neuroprotection and anticholinesterase activity were fully evaluated. The anti-amyloid aggregation, antioxidant and metal-chelation activities of the best compound were also investigated by standard methods to find a new multi-functional agent against AD. Results The primary biological screening demonstrated that all compounds had significant neuroprotection activity against H2O2-induced cell damage in PC12 cells. Compound 19 as the most potent butyrylcholinesterase (BuChE) inhibitor (IC50 = 7.55 μM) having significant neuroprotection activity as level as reference drug was selected for further biological evaluations. Docking and kinetic studies revealed non-competitive mixed-type inhibition of BuChE by compound 19. It could significantly reduce the formation of the intracellular reactive oxygen species (ROS) and showed excellent reducing power (85.57 mM Fe+2), comparable with quercetin and lipoic acid. It could also moderately inhibit Aβ aggregation and selectively chelate with copper ions in a 2:1 M ratio.

Conclusion Compound 19 could be considered as a hopeful multifunctional agent for the further development gainst AD owing to the acceptable neuroprotective and anti-BuChE activity, moderate anti-Aβ aggregation activity, outstanding antioxidant activity as well as selective copper chelation ability.

Leili Jalili-Baleh1 & Hamid Nadri2 & Hamid Forootanfar3 & Tuba Tüylü Küçükkılınç4 & Beyza Ayazgök4 & Mohammad Sharifzadeh5 & Mahban Rahimifard6 & Maryam Baeeri6 & Mohammad Abdollahi6 & Alireza Foroumadi1 & Mehdi Khoobi

Biomaterials Group, Pharmaceutical Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran 1417614411, Iran 2 Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran 3 Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences, Kerman, Iran 4 Faculty of Pharmacy, Department of Biochemistry, Hacettepe University, Ankara, Turkey 5 Department of Pharmacology and Toxicology, Faculty of Pharmacy, Toxicology, and poisoning Research Centre, Tehran University of Medical Sciences, Tehran, Iran 6 Toxicology and Diseases Group, Pharmaceutical Sciences Research Center (PSRC), The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences (TUMS), Tehran, Iran

Introduction 

Alzheimer’s disease (AD), a multifaceted and irreversible neurodegenerative disease, is the major cause of dementia, and one of the biggest challenges of current pharmaceutical research [1]. During the past decades, despite substantial efforts to discover the pathogenic mechanisms of Alzheimer's Disease, the exact etiology of this disease is still complex. The pathological factors playing a remarkable role in the development of AD are deficiency of brain cholinergic neurotransmitters level, accumulation of senile plaques particularly as extracellular β-amyloid (Aβ) deposits, hyperphosphorylated τ-protein aggregation [2], dyshomeostasis of biometals, oxidative stress, and neuroinflammation [3]. Notwithstanding all attempts performed to find innovative solutions to control the hallmarks of the disease like gene and stem cell therapy, this kind of therapy are still a prospective method for AD treatment and there are still many unsolved problems before they can be used in clinical applications [4, 5]. The currently available therapeutic method is mostly based on the increasing cholinergic neurotransmission, through acetylcholinesterase inhibitors (AChEI). Like AChE, butyrylcholinesterase (BuChE) as a coregulator of the ACh degradation, is an important enzyme involved in the neurotransmission [6]. The role of BuChE in AD pathology is unknown, but in vivo studies revealed that BuChE associated with Aβ plaques has a key role in AD plaque maturation [7, 8]. To complete the symptomatic approaches, it is necessary to target simultaneously multiple pathological processes involved in AD [9, 10]. 

Click to Cistanche DHT for Alzheimer's disease

Currently, the multi-target directed ligand (MTDL) strategy has been completely accepted as the main strategy for the drug design and discovery in AD, which is based on a combination of appropriate pharmacophoric groups in one molecule providing effective pharmacological responses for various potential receptors or enzymatic targets [11]. In recent years, many potential multifunctional agents have been designed based on MTDLs strategy against AD [12–16]. Moreover, quite an amount of natural-origin products is being used either as marketed pharmaceuticals or as bioactive molecular fragments in the development of hybrid drugs to combat the above-referred major risk factors involved in the pathogenesis of AD [17]. Chromones are ubiquitously found in plants and are known as phytochemicals with a benzo-γ-pyrone structure, which possesses multiple ranges of pharmacological effects [18]. Recent studies revealed that chromone-based compounds were effective against dementia through free radicals scavenging and metal ions chelating [19–22]. Chromones have also showed anticholinesterase [23, 24], neuroprotective [25, 26], anti-inflammatory properties [27], and could effectively disrupt amyloid-β aggregation [28, 29]. Fernandez-Bachiller and co-workers introduced tacrinechromone hybrids having improved ChE inhibitory, as well as antioxidant, inactivation compared to tacrine [30]. Lipoic acid (LA), as a naturally occurring antioxidant in animals, humans, and plants acts as an essential cofactor in many biochemical pathways [31]. A diverse range of pharmacological properties has been reported about this organosulfur. It can control the pathogenesis or progression of AD by increasing the level of acetylcholine as well as decreasing oxidative stress, inflammation, and Aβ plaque formation [32–34]. Apocrine is a hybrid of LA and tacrine introduced by Rosini as an effective drug candidate against AD due to its multiple biological properties, such as AChE and BChE inhibition activity, inhibition of AChE-induced Aβ aggregation, and cell protection against ROS [35]. 

G. Nesi et al. combined rivastigmine with LA and chromone. They found that the combination led to the addition of anti-oxidants and anti-amyloid aggregating properties of LA and chromone scaffolds to the anticholinesterase activity of rivastigmine as the currently used drugs, and resulted in the formation of the multifunctional compound to treat AD [28]. Based on the above findings, chromone scaffold is an important pharmacophore having a great role in ChEs inhibition with antioxidant and metal chelation activities. LA is also the promising lead structure improving BuChE inhibition activity, neuroprotection potency, anti-amyloid aggregation, antioxidant, and metal chelation activities (Fig. 1). These unique characteristics of chromone and LA have made them promising scaffolds to develop novel multi-target compounds having improved therapeutic efficacy in the treatment of different neurodegenerative disorders, especially AD [36–42]. Considering the beneficial biological effects of chromone and LA scaffolds and the efficacy of triazole nucleus as an attractive pharmacophore to connecting various therapeutically active agents [43–45], in this work, we encouraged to conjugate these valuable pharmacophores to each other and evaluate their efficacy against various targets. Two series of chromone derivatives conjugated with LA were synthesized via click reaction (Fig. 1, compounds 9–17 and 18–20) and the neuroprotective activity and ChEs inhibition activity of all the compounds were evaluated. The antioxidant, metal chelation, and anti-amyloid activities of the selected compound were also evaluated. To the best of our knowledge, this is the first work reporting the synthesis of chromone-LA hybrids and their biological activities against AD.

Results and discussion 

Chemistry As illustrated in Scheme 1, compound 3 was initially synthesized via reaction of substituted 2′-hydroxy acetophenone with 4-methoxy benzaldehyde through a domino aldol-Michael-oxidation reaction catalyzed by pyrrolidine and iodine in dimethyl sulfoxide (DMSO). Compound 3 was converted to the corresponding 2-(4-hydroxyphenyl)-4H-chrome-4-one derivatives (4a-d) by BBr3 in dichloromethane. Compound 5 was also prepared in one step from the reaction of 2,4- dihydroxyacetophenone and triethyl orthoformate in the presence of 70% perchloric acid leading to the formation of perchlorate salt which was then hydrolyzed to reach the target compound (5). The bromoalkoxy intermediates 6a-i and 8a-c were then prepared via the reaction of 4a-d or 5 with the appropriate amount of dibromoalkanes in an acetone solution of anhydrous K2CO3 under refluxing condition for 4 h. Compound 7 was separately synthesized through the amidation reaction between LA and propargylamine in the presence of 4- dimethyl aminopyridine (DMAP) and N, N ′ - dicyclohexylcarbodimide (EDCI). 

Target compounds 9–20 were finally prepared through a one-pot three-component reaction between compounds 6a-i or 8a-c, sodium azide, and compound 7 catalyzed by copper (II). Biological assay Primarily, neuroprotection and cholinesterase inhibitory activities of all target compounds 9–20 were evaluated to find the best potent compound for further studies. Neuroprotection potency against PC12 cell damage induced by H2O2 Oxidative damage and neurotoxicity created by H2O2 is considered as the most important factor controlling the progress of neurodegenerative disorder [60]. Therefore, neuroprotection activity of the prepared chromone–LA conjugates 9–20 were screened at different concentrations of 1, 5, 10, 20, and 50 μM using MTT assay. All compounds, in all concentrations, could significantly increase the cell viability of PC12 cells dose-dependently, even at a low concentration of 1 μM (Table 1, p < 0.001). 

Interestingly, compounds 15, 16, and 17 having five-carbon chain lengths (n = 5) exhibited higher neuroprotective activity than that of quercetin as a reference drug in all concentrations. By comparison between compounds 9, 11, and 15 bearing simple 2-phenyl-4H-chromene-4-one moiety and different carbon chain length (n = 3, 4, and 5 for compounds 9, 11, and 15, respectively), it could be implied that increasing the size of cross-linker could increase the neuroprotection activity of the target compounds (Table 1). The same behavior was also seen for compounds 18–20 as 4H-chromene-4-one derivatives substituted at 7 positions. Notably, the unsubstituted chrome derivatives had less neuroprotective activity on the H2O2-induced cell death than halosubstituted analogs (compare compound 9 with compound 10, compound 11 with compounds 12–14, and compound 15 with compounds 16 and even 17 at especially high concentrations). Cholinesterase inhibitory activity AChE and BuChE inhibitory activity of all chromone–LA conjugates 9–20 were investigated. Table 2 shows the IC50 values of the compounds compared with standard drug donepezil. No significant effect was observed against AChE at 100 μM for most of the compounds, except 7-fluoro derivatives (13) with moderate activity (IC50 = 56.50 μM). The results revealed that the activity of the 7- position substituted derivatives (18–20) against BuChE was better than the 2-position modified derivatives (9–17). 

Among the 7-substituted derivatives, only compounds 18 and 19 with 3 or 4 atom chain lengths showed appropriate anti-BuChE activity (IC50 = 15.32 and 7.55 μM, respectively). Compound 20 with 5 carbon spacer (n = 5) revealed no activity against BuChE confirming the great effect of the cross-linker and size of the molecule to occupy the enzyme active site. Therefore, the extension of the linker has no positive effect on the BuChE inhibitory potency. When tacrin bearing halogen substituent was conjugated to LA by 3 carbon spacer, the AChE, and BuChE inhibition activities, especially AChEI activity, were improved in comparison with tacrin [35]. The study confirmed the important role of the length of the cross-linker on ChEs inhibition activity of the target compounds. A previous study also revealed that hybridization of tacrin with chromone scaffold improves BuChE inhibition activity more than that of AChEI which is in good agreement with the achievement of our results [30]. The same result was also observed when other well-known pharmacophores were combined. Attachment of LA and/or chromone to rivastigmine resulted in a higher BuChE inhibition activity than AChE inhibition [28]. It seems that LA can improve the BuChE inhibition activity of the mother scaffold more than AChE inhibition activity. Based on the results, it seems that electron-donating groups on chromone scaffold and also using an appropriate spacer to prevent molecular folding may facilitate proper interactions with the enzyme transforming target compounds into more potent inhibitors.